|KOROBOVA, OLGA - State Research Center For Applied Microbiology And Biotechnology|
|SCHISCHKOVA, NINA - State Research Center For Applied Microbiology And Biotechnology|
|KISELEVA, NATALIA - State Research Center For Applied Microbiology And Biotechnology|
|KOPYLOV, PAUL - State Research Center For Applied Microbiology And Biotechnology|
|PRYAMCHUK, SERGEY - State Research Center For Applied Microbiology And Biotechnology|
|ABAEV, IGOR - State Research Center For Applied Microbiology And Biotechnology|
Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2012
Publication Date: 12/31/2012
Citation: Schmelcher, M., Korobova, O., Schischkova, N., Kiseleva, N., Kopylov, P., Pryamchuk, S., Donovan, D.M., Abaev, I. 2012. Staphylococcus haemolyticus prophage endolysin relies on CHAP endopeptidase lytic activity for lysis from without. FEMS Microbiology Letters. 162(2-3):289-298.
Interpretive Summary: Bovine mastitis results in annual costs between $1.7 billion and $2 billion in the United States only. One of the most relevant causative agents of mastitis is Staphylococcus aureus. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Furthermore, multi-drug resistant S. aureus strains (MRSA) are becoming increasingly prevalent in clinical settings. Therefore, alternative antimicrobials for the control of this pathogen are needed. In this study, the Staphylococcus haemolyticus JCSC1435 prophage FSH2 endolysin (Haemo lysin) was purified and shown to exhibit lytic activity against several live staphylococcal strains in three different in vitro assays. Truncation analysis revealed that the CHAP domain of the Haemo lysin contributes most to its lytic activity, and that the SH3b cell wall binding domain is required for the highest activity of the full-length enzyme. Furthermore, we demonstrate that bivalent metal ions can significantly increase lytic activity. Contribution of Accomplishment to Solving the Problem-- These findings provide new evidence that bacteriophage endolysins have a high potential as alternative antimicrobials for the control of pathogenic bacteria such as S. aureus. It is widely accepted that, as opposed to antibiotics, the chance of resistance development against phage endolysins is low. Despite efforts to find them, bacterial strains resistant to phage lysins have not been reported yet. Previous studies have shown that fusion of functional modules from different peptidoglycan hydrolases yields enzymes with new and potentially optimized properties. To this end, truncation analysis as performed in this study is important for identification of potent domains.
Technical Abstract: Staphylococcus aureus is an important human pathogen, with multi-drug resistant strains (methicillin-resisant S. aureus; MRSA) becoming increasingly prevalent, in both human and veterinary clinics. S. aureus bovine mastitis has a major impact on the dairy industry with annual losses reported several years ago to be between $1.7 billion and $2 billion in the United States alone. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a new promising source of antimicrobials against these pathogens. The endolysin of prophage FSH2 of Staphylococcus haemolyticus strain JCSC1435 (Haemo lysin) is a peptidoglycan hydrolase consisting of two enzymatically active domains (CHAP endopeptidase and amidase) and a C-terminal SH3b cell wall binding domain. In this work, we recombinantly expressed the Haemo lysin in E. coli and demonstrate its lytic activity against live S. aureus and S. epidermidis cells. A series of deletion mutants was constructed in order to investigate the contribution of each functional module to bacterial lysis from without, using zymogram analysis, plate lysis, and turbidity reduction assays. A truncation protein only comprising the CHAP domain retained considerable lytic activity, and this activity was not enhanced by fusion to the C-terminal SH3b domain. On the contrary, the amidase domain was only very weakly active in the truncations, suggesting that the Haemo lysin mainly relies on CHAP activity for lysis from without. The full length Haemo lysin was shown to exhibit four times higher lytic activity than a construct containing both enzymatic domains but lacking the SH3b domain. Furthermore, we investigated the influence of bivalent metal ions on lytic activity of all constructs. The addition of 1 mM Mg2+, Mn2+, and Ca2+ resulted in an increase in activity of up to 60%. Overall, our results demonstrate the high potential of phage endolysins such as the Haemo lysin as sources of antimicrobials for the treatment of S. aureus infections.